Spatiotemporal Analysis of Mesenchymal Stem Cells Fate Determination by Inflammatory Niche Following Soft Tissue Injury at a Single-Cell Level

Heterotopic ossification (HO), often arising in response to traumatic challenges, results from the aberrant osteochondral differentiation of mesenchymal stem cells (MSCs). Nevertheless, the impact of trauma-induced inflammatory exposure on MSC fate determination remains ambiguous. In this study, the cellular diversity within inflammatory lesions is elucidated, comprising MSCs and several innate and adaptive immune cells. It is observed that quiescent MSCs transition into cycling MSCs, subsequently giving rise to chondrogenic (cMSC) and/or osteogenic (oMSC) lineages within the inflammatory microenvironment following muscle or tendon injuries, as revealed through single-cell RNA sequencing (scRNA-seq), spatial transcriptome and lineage tracing analysis. Moreover, these investigations demonstrate that neutrophils and natural killer (NK) cells enhance transition of quiescent MSCs into cycling MSCs, which is also controlled by M1 macrophages, a subpopulation of macrophages can also stimulate cMSC and oMSC production from cycling MSCs. Additionally, M2 macrophages, CD4+ and CD8+ T lymphocytes are found to promote chondrogenesis. Further analysis demonstrates that immune cells promotes the activation of signaling transducers and activators of transcription (STAT) pathway and phosphoinositide 3 (PI3K)/protein kinase B (AKT) pathway in MSC proliferation and osteochondral progenitors' production, respectively. These findings highlight the dynamics of MSC fate within the inflammatory lesion and unveil the molecular landscape of osteoimmunological interactions, which holds promise for advancing HO treatment. The dynamics of mesenchymal stem cell (MSC) lineage specification and associated dynamic regulation is highlighted by immune cells during HO formation, i.e., an elaborate trajectory of MSCs is characterized during aberrant osteochondrogenesis and demonstrates that MSCs proliferation are regulated by M1 macrophages (M1), neutrophils, and natural killer (NK) cells via janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and differentiation process, including chondrogenic MSC (cMSC) and osteogenic MSC (oMSC) production are determined by M2 macrophages (M2), CD4+ and CD8+ T cells via phosphoinositide 3 kinase (PI3K) /protein kinase B (AKT) signaling activation. image